Biologically active polypeptides or proteins which are attached to insoluble carrier materials, such as polymeric particles, have been used in a variety of ways. For example, the diagnosis of pathological or other conditions in human beings and animals is often carried out using immunological principles for the detection of an immunologically reactive species, for example antibodies or an antigen, in the body fluids of the person or animal. An antigen is generally known as a foreign substance, such as a drug, hapten, toxin, lectin, polypeptide or protein which, when introduced into the body, causes the production of certain soluble proteins known as antibodies.
Other proteins and amine-containing compounds, such as enzymes, avidin, biotin or polysaccarides, have been covalently linked to various carrier materials for use in affinity chromatography, enzymatic reactions, specific binding reactions and immunoassays. Among useful carrier materials are sheep and human erythrocytes, bacterial cells, latex particles, resinous particles and finely divided diazotized amino cellulose. For example, carrier particles prepared from sparingly water-soluble monomers (such as epoxy group-containing monomers) in the absence of emulsifiers are described in U.S. Pat. No. 4,415,700 (issued Nov. 15, 1983 to Batz et al). Other compounds, such as diamines, dihydrazides, mercaptoalkylamines and dimercaptans have been attached to carrier materials as linking moieties for later attachment of drugs, enzymes or other reactive species.
Carboxylated latex particles have also been used to prepare diagnostic reagents as described, for example, in U.S. Pat. No. 4,181,636 (issued Jan. 1, 1980 to Fischer). As described therein, the standard procedure for covalently attaching an immunologically reactive species to the particles having surface carboxyl groups involves the use of a water-soluble carbodiimide. While producing useful reagents, this procedure tends to activate the exposed reactive groups of the reactive species as well as the carboxyl groups. The result is intramolecular and intermolecular crosslinking or polymerization of the immunologically reactive species, and a significant portion of the species is thus impaired from complexation with a receptor molecule. Because the reactive species, for example an antibody, is usually very costly, this problem represents a serious economic loss. It has also been evident that the use of carbodiimides to attach proteins to carrier particles is not as efficient as desired at certain protein levels.
An important advance in the art was achieved with the use of carbamoylonium compounds which allows for rapid and more efficient attachment of biological compounds to carboxylated particles. The carbamoylonium compounds provide minimal crosslinking or deactivation of the reactive amino or sulfhydryl groups.
This advance is described in considerable detail in EP-A-O 308 235 (published Apr. 26, 1989, corresponding to U.S. Ser. No. 373,304, filed Jun. 29, 1989 by Sutton, Danielson, Bagchi and Scensny as a of U.S. Ser. No. 286,097, filed Dec. 19, 1988 which in turn is of U.S. Ser. No. 098,429, filed Sep. 18, 1987). The compounds are generically described in the noted reference. More particularly, pyridinium salts are described having piperidinylcarbonyl, piperazinylcarbonyl or morpholinylcarbonyl groups attached thereto. Named compounds are 1-(4-morpholinocarbonyl)-4-(2-sulfoethyl)pyridinium hydroxide, inner salt, and 1-(4-morpholinocarbonyl)pyridinium chloride.
It has been found, however, that when the noted carbamoylonium compounds are used to attach proteins to carboxylated polymer particles, they generate strongly nucleophilic amine by-products (such as morpholine) in the course of the activation reactions. These by-products compete with the proteins for activated carboxylic sites on the particles, and tend to terminate attachment of proteins as sites are used up quickly. Thus, activation with the noted carbamoylonium compounds, such as 1-(4-morpholinocarbonyl)-4-(2-sulfoethyl)-pyridinium hydroxide, inner salt, is not very efficient.
It would be desirable to have more efficient activating agents which do not generate undesirable by-products.